The invention relates to DNA sequences for human matrix metalloproteases as well as to homologous sequences derived therefrom. It furthermore relates to the proteins and protein variants, coded by the DNA sequences, their expression, preparation and utilization. Areas of application are molecular biological, medical and pharmaceutical research, medical diagnosis and therapy and the pharmaceutical and biotechnological industry.
Matrix metalloproteases hydrolyze proteins of the extracellular matrix. They change the matrix structure and effect cell-matrix interactions. The matrix metalloproteases include collagenases, gelatinases, stromelysins and metalloelastases [1]. The following are some of the physiological processes, in which the enzymes participate: ovulation [2], embryo implantation in the uterus [3], cell migrations and tissue inversions during embryo genesis [4], involution of the mammary gland [5] and of the uterus [6] and angiogenesis [7]. Matrix metalloproteases play an important role in wound healing and scar formation [8], in metastasizing of tumors cells [9, 10], in rheumatic arthritis and osteoarthritis [11, 12] and in periodontal diseases [13].
All matrix metalloproteases contain a Zn2+ ion in the active center. The activation of the matrix metalloproteases, synthesized in the form of inactive pro-enzymes, requires the dissolution of a bond between the Zn2+ ion in the active center and a Cys group in the it-terminal propeptide of matrix metalloproteases (cysteine switch) [14]. Matrix metalloproteases consist of several protein domains, which exhibit homology among members of the Protease family [1, 14]. Whereas the protease matrilysin consists only of a propeptide and of the amino acid sequence of the catalytic domain, other matrix metalloproteases contain, in addition, a hemopexin-like sequence of about 200 amino acids. The gelatinases A and B contain additional amino acid sequences. Known human matrix metalloproteases, their molecular weights and their preferred substrates are listed in Table 1.
The different matrix metalloproteases are distinguished not only by a characteristic, macromolecular specificity for matrix proteins. Their activity is controlled on different molecular and cellular levels:
1. Regulation of the synthesis of matrix metalloproteases by growth factors, cytokines, polypeptide hormones, prostaglandins, glucocorticoids, estrogen, progesterone and other effectors [1, 14].
2. Binding of matrix metalloproteases to membrane a receptors [15].
3. Activation of inactive proenzymes by specific hydrolysis of the respective propeptides [16] or by oxidation [17].
4. Inhibition of matrix metalloproteases by specific protein inhibitors such as TIMP-1, TIMP-2 and TIMP-3 (TIMP=Tissue Inhibitor of Matrix Metalloproteases) [16].
5. Proteolytic degradation of matrix metalloproteases.
Matrix metalloproteases are being investigated intensively because of their important physiological functions and their role in the pathogenesis of diseases. There is interest in finding and characterizing further matrix metalloproteases.
It is an object of the present invention to make accessible novel and previously unknown human matrix metalloproteases for medical research, diagnosis and therapy. The task consists of identifying and isolating DNA sequences for matrix metalloproteases and of characterizing the proteins coded by the DNA sequences.
Novel matrix metalloproteases are discovered by the following method. In sequences of known matrix metalloproteases, conserved amino acid sequences are selected. Two suitable sequences are amino acids about a conserved Cys group in the propeptide (cysteine switch) and amino acids, which participate in the Zn2+ binding in the active center of the enzymes. Oligonucleotides are synthesized for the selected peptides. Polymerase chain reactions (PCR) are carried out with the oligonucleotides and cDNA, which can be obtained by reverse transcription of mRNA from cells and tissue. Synthesized DNA fragments are cloned and sequenced. The sequences determined are compared with sequences in gene data banks. PCR products with novel, previously unknown nucleotide sequences and homologous with DNA sequences of matrix metalloproteases are used as probes for determining the gene expression and for obtaining complete cDNA sequences from cDNA libraries. The nucleotide sequences of complete cDNA are determined. The amino acid sequences of the corresponding proteins are derived by translation of the coding nucleotide regions and analyzed by known methods.